Chromosome 16 inversion, inv(16), is one of the most common chromosome abnormalities in human acute myeloid leukemia (AML). A fusion gene between the core binding factor B (CBFB) gene and the myosin heavy chain 11 (MYH11) gene is generated by this inversion. CBFB encodes a transcription factor while MYH11 encodes the smooth muscle form of myosin heavy chain (SMMHC). Using a mouse knock-in strategy, we showed that the fusion gene Cbfb-MYH11 is necessary but not sufficient for leukemogenesis. Last year we reported the identification of genes that specifically cooperate with Cbfb-MYH11 in leukemogenesis. Neonatal injection of Cbfb-MYH11 knock-in chimeric mice with retrovirus 4070A led to the development of AML in 2-5 months. Each leukemia sample contained one or a few viral insertions, suggesting that alteration of one gene could be sufficient to synergize with Cbfb-MYH11. The chromosomal position of 67 independent retroviral insertion sites (RIS) was determined, and 90% of the RIS mapped within 10 kb of a flanking gene. In total 54 candidate genes were identified, 6 of them were common insertion sites. This is the first retroviral genetic screen to identify genes that cooperate with a fusion gene linked to the pathogenesis of human leukemia. In order to study the function of Cbfb gene in T cell development, we used a mouse line with floxed exons 5 and 6 of Cbfb inserted 5? to the Cbfb-MYH11 fusion cassette, which produced pseudo-normal mice (loxKI). By crossing the loxKI mice with mice expressing the Cre gene under the control of the T cell-specific Lck promoter (LckCre), we generated LckCre-loxKI double positive mice, in which the floxed exon 5 and 6 were deleted and Cbfb-MYH11 re-expressed only in the thymus when Lck started to express. The LckCre-loxKI mice were viable. However, their thymic development was severely impaired: The size of the thymuses in the mutant mice was about half the normal size, and the total number of thymocytes in the mutant mice was 10-20-fold reduced. FACS analysis of thymocytes from 4 to 12 week old mice showed a developmental blockade at the CD4/CD8-double negative (DN) stage, which was characterized by lower percentage of double positive cells and higher percentage of double negative cells. In addition, the CD4 : CD8 ratio was altered. Furthermore, the mature T cell population size in the spleen of the mutant mice was lower than that of the control mice. Our preliminary data suggested that Cbfb plays an important role in T cell development. The mechanism through which Cbfb affects the T cell development is currently under investigation. It is likely that the phenotype reflects the combined effect of missing all three Runx genes, since the phenotype described here is more severe than either Runx1 or Runx3 null alone. Chromosomal rearrangements affecting RUNX1 and CBFB are common in acute leukemias. These mutations result in the expression of fusion proteins that act in a dominant negative manner to suppress the normal function of the CBFb/RUNX1 complex. In addition, loss-of-function mutations in RUNX1 have been identified in sporadic cases of acute myeloid leukemia (AML) and in association with familial platelet disorder with propensity to develop AML (FPD/AML). In order to examine the hypothesis that decreased gene dosage of RUNX1 may be a critical event in the development of leukemia, we treated chimeric mice generated from Runx1-/- embryonic stem (ES) cells that have homozygous disruption of the RUNX1 gene, as well as Runx1+/- mice with N-ethyl-N-nitrosurea (ENU). The heterozygous Runx1+/- mice did not show increased incidence of any malignancy. On the other hand, we observed an increased incidence of precursor T-lymphoblastic lymphoma in Runx1-/- compared to wild-type chimeras, and confirmed that the tumors were of ES cell origin. It was determined by PCR that Runx1-/- ES cells contributed to the T cell progenitor population in the chimeras prior to leukemia development, which may explain the tissue-specificity of the malignancy we observed. Our results suggest that deficiency of Runx1 can indeed predispose mice to hematopoietic malignancies.